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Abstract:

This invention describes a process for separating a fusion protein or
polypeptide in the form of its precursor from a mixture containing said
fusion protein and impurities, which comprises contacting said fusion
protein with a resin containing immobilized metal ions, said fusion
protein covalently operably linked directly or indirectly to an
immobilized metal ion-affinity peptide, binding said fusion protein to
said resin, and selectively eluting said fusion protein from said resin.

Claims:

1. A polypeptide, protein or protein fragment represented by the formula
R1-Sp1-(His-Z1-His-Arg-His-Z2-His)-Sp2-R2,
wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal
ion-affinity peptide, R1 is hydrogen, a polypeptide, protein or
protein fragment, Sp1 is a covalent bond or a spacer comprising at
least one amino acid residue, R2 is hydrogen, a polypeptide, protein
or protein fragment, Sp2 is a covalent bond or a spacer comprising
at least one amino acid residue, Z1 is an amino acid residue
selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Ile, Lys,
Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is an amino acid residue
selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly,
Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val.

2. The polypeptide, protein or protein fragment of claim 1, wherein
Z1 is selected from the group consisting of Ala, Asn, Ile, Lys, Phe,
Ser, Thr, and Val, and Z2 is selected from the group consisting of
Ala, Asn, Gly, Lys, Ser, Thr and Tyr.

3. The polypeptide, protein or protein fragment of claim 1, wherein
Z1 and Z2 are selected from the group consisting of: (a)
Z1 is Asn and Z2 is Gly; (b) Z1 is Asn and Z2 is Lys
(c) Z1 is Lys and Z2 is Gly. (d) Z1 is Lys and Z2 is
Lys. (e) Z1 is Ile and Z2 is Asn; (f) Z1 is Thr and
Z2 is Ser; (g) Z1 is Ser and Z2 is Tyr; (h) Z1 is Val
and Z2 is Ala; and (i) Z1 is Ala and Z2 is Lys.

4. The polypeptide, protein or protein fragment of claim 1, wherein
R1 or R2 is hydrogen.

5. The polypeptide, protein or protein fragment of claim 1, wherein
R1 or R2 is an amino acid residue.

6. The polypeptide, protein or protein fragment of claim 1, wherein
Sp1 or Sp2 is a spacer comprising a proteolytic cleavage site,
a fusion protein, a secretion sequence, a leader sequence for cellular
targeting an antibody epitope or an internal ribosomal sequences.

7. The polypeptide, protein or protein fragment of claim 1, wherein
Sp1 or Sp2 is a spacer comprising a proteolytic cleavage site.

8. The polypeptide, protein or protein fragment of claim 7, wherein the
proteolytic cleavage site is cleaved with enterokinase.

9. The polypeptide, protein or protein fragment of claim 1, wherein any
one of Sp1, Sp2, R1 and R2 comprises at least one of
the amino acid sequences selected from the group consisting of SEQ ID
NOS: 1-17.

10. The polypeptide, protein or protein fragment of claim 1, wherein
Sp1 or Sp2 is a spacer comprising the enzyme
glutathione-S-transferase of the parasite helminth Schistosoma japonicum.

where: D, Y and K are their representative amino acids; X20 and
X21 are independently a hydrogen or a covalent bond; each X1
and X4 is independently a covalent bond or at least one amino acid
residue selected from the group consisting of aromatic amino acid
residues and hydrophilic amino acid residues; each X2, X3,
X7 and X8 is independently an amino acid residue selected from
the group consisting of aromatic amino acid residues and hydrophilic
amino acid residues; X5 is a covalent bond or a spacer domain, the
spacer domain comprising at least one amino acid or a combination of
multiple or alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X)m--, wherein m is 1 to 6 and X is selected
from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X9 is a
covalent bond or an aspartate residue; and n is 0, 1 or 2.

where: D, Y, K are their representative amino acids; X20 and
X21 are independently a hydrogen or a covalent bond; each X2,
X3, X7 and X8 is independently an amino acid residue
selected from the group consisting of aromatic amino acid residues and
hydrophilic amino acid residues; X5 is a covalent bond or a spacer
domain, the spacer domain comprising at least one amino acid or a
combination of multiple or alternating histidine residues, said
combination comprising His-Gly-His, or -(His-X)m--, wherein m is 1
to 6 and X is selected from the group consisting of Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr
and Val; X9 is a covalent bond or an aspartate residue; and n is at
least 2.

where: D, Y, and K are their representative amino acids; X20 and
X21 are independently a hydrogen or a covalent bond; X10 is a
covalent bond or an amino acid; each X2, X3, X7 and
X8 is independently an amino acid residue selected from the group
consisting of aromatic amino acid residues and hydrophilic amino acid
residues; X5 is a covalent bond or a spacer domain, the spacer
domain comprising at least one amino acid or a combination of multiple or
alternating histidine residues, said combination comprising His-Gly-His,
or -(His-X)m--, wherein m is 1 to 6 and X is selected from the group
consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; X9 is a covalent bond or
an aspartate residue; and n is at least 2.

where: D, Y and K are their representative amino acids; X20 and
X21 are independently a hydrogen or a covalent bond; each X11
is a covalent bond or an amino acid; each X12 is an amino acid
selected from the group consisting of aromatic amino acid residues and
hydrophilic amino acid residues; each X13 is a covalent bond or at
least one amino acid selected from the group consisting of aromatic amino
acid residues and hydrophilic amino acid residues; X14 is a covalent
bond or a spacer domain, the spacer domain comprising at least one amino
acid or alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X)m--, wherein m is 1 to 6 and X is selected
from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X15 is a
covalent bond or an aspartate residue; and n is 0 or at least 1.

18. A polypeptide, protein or protein fragment represented by the formula
R1-Sp1-(His-Z1-His-Arg-His-Z2-His)t-Sp2-R.s-
ub.2, wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide, t is at least 2, R1 is hydrogen, a
polypeptide, protein or protein fragment, Sp1 is a covalent bond or
a spacer comprising at least one amino acid residue, R2 is hydrogen,
a polypeptide, protein or protein fragment, Sp2 is a covalent bond
or a spacer comprising at least one amino acid residue, Z1 is an
amino acid residue selected from the group consisting of Ala, Arg, Asn,
Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is
an amino acid residue selected from the group consisting of Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and
Val.

19. The peptide of claim 18, wherein Z1 and Z2 are selected
from the group consisting of: (a) Z1 is Asn and Z2 is Lys; and
(b) Z1 is Lys and Z2 is Gly.

20. A polypeptide, protein or protein fragment represented by the formula
R1-Sp1-[(His-Z1-His-Arg-His-Z2-His)-Sp2]t-R-
2, wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide, t is at least 2, R1 is hydrogen, a
polypeptide, protein or protein fragment, Sp1 is a covalent bond or
a spacer comprising at least one amino acid residue, R2 is hydrogen,
a polypeptide, protein or protein fragment, Sp2 is a covalent bond
or a spacer comprising at least one amino acid residue, Z1 is an
amino acid residue selected from the group consisting of Ala, Arg, Asn,
Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is
an amino acid residue selected from the group consisting of Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and
Val; and each Sp2 of the recombinant polypeptides, proteins or
protein fragments may be the same or different.

21. The peptide of claim 20, wherein Z1 and Z2 are selected
from the group consisting of: (a) Z1 is Asn and Z2 is Lys; and
(b) Z1 is Lys and Z2 is Gly.

Description:

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.
10/460,524, filed Jun. 12, 2003, which is a non-provisional application
claiming priority from provisional Application Ser. No. 60/388,059, filed
Jun. 12, 2002, the content of each of which is hereby incorporated herein
by reference.

FIELD OF THE INVENTION

[0002] This invention relates to affinity peptides, fusion proteins
containing affinity peptides, genes coding for such proteins, expression
vectors and transformed microorganisms containing such genes, and methods
for the purification of the fusion proteins.

BACKGROUND OF THE INVENTION

[0003] The possibility of preparing hybrid genes by gene technology has
opened up new routes for the analysis of recombinant proteins. By linking
the coding gene sequence of a desired protein to the coding gene sequence
of a protein fragment having a high affinity for a ligand (affinity
peptide), it is possible to purify desired recombinant proteins in the
form of fusion proteins in one-step using the affinity peptide.

[0004] Immobilized metal affinity chromatography (IMAC), also known as
metal chelate affinity chromatography (MCAC), is a specialized aspect of
affinity chromatography. The principle behind IMAC lies in the fact that
many transition metal ions, e.g., nickel, zinc and copper, can coordinate
to the amino acids histidine, cysteine, and tryptophan via electron donor
groups on the amino acid side chains. To utilize this interaction for
chromatographic purposes, the metal ion is typically immobilized onto an
insoluble support. This can be done by attaching a chelating group to the
chromatographic matrix. Most importantly, to be useful, the metal of
choice must have a higher affinity for the matrix than for the compounds
to be purified.

[0005] In U.S. Pat. No. 4,569,794, Smith et al. disclose the preparation
of a fusion protein containing a metal ion-affinity peptide linker and a
biologically active polypeptide, expressing the fusion protein, and
purifying it using immobilized metal ion chromatography. Because
essentially any biologically active polypeptide could be used, this
approach enabled the convenient expression and purification of
essentially biologically active polypeptide by immobilized metal ion
chromatography.

[0007] One aspect of the present invention is a peptide which is
relatively hydrophilic, is capable of exhibiting appropriate biological
activity, and has a relatively high affinity for coordinating metals.
Advantageously, this metal ion-affinity peptide may be incorporated into
a fusion protein to enable ready purification of the fusion protein from
aqueous solutions by immobilized metal affinity chromatography. In
addition to the metal ion-affinity peptide, the fusion protein typically
comprises a protein or polypeptide of interest, covalently linked,
directly or indirectly, to the metal ion-affinity peptide.

[0008] Briefly, therefore, the present invention is directed to a peptide
represented by the formula
R1-Sp1-(His-Z1-His-Arg-His-Z2-His)-Sp2-R2,
wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal
ion-affinity peptide, R1 is hydrogen, a polypeptide, protein or
protein fragment, Sp1 is a covalent bond or a spacer comprising at
least one amino acid residue, R2 is hydrogen, a polypeptide, protein
or protein fragment, Sp2 is a covalent bond or a spacer comprising
at least one amino acid residue, Z1 is an amino acid residue
selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile,
Lys, Phe, Pro, Ser, Thr, Trp, and Val; and Z2 is an amino acid
residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val.

[0009] The present invention is further directed to a process for
separating a recombinant protein or polypeptide from a liquid mixture
wherein the recombinant protein or polypeptide comprises a metal
ion-affinity peptide having the sequence
His-Z1-His-Arg-His-Z2-His (SEQ ID NO: 24) and Z1 and
Z2 are as previously defined. In the process, the mixture is
combined with a solid support having immobilized metal ions to bind the
recombinant protein or polypeptide, and eluting the fusion protein from
the solid support.

[0010] The present invention is further directed to vectors and host cells
for recombinant expression of the nucleic acid molecules described
herein, as well as methods of making such vectors and host cells and for
using them for production of the polypeptides or peptides of the present
invention by recombinant techniques.

[0011] The present invention is further directed to a kit for the
expression and/or separation of the recombinant proteins or polypeptides
from a mixture wherein the recombinant proteins or polypeptides contain
the sequence
R1-Sp1-(His-Z1-His-Arg-His-Z2-His)-Sp2-R2,
and R1, R2, Sp1, Sp2, Z1 and Z2 are as
previously defined. The kit may comprise, in separate containers, the
nucleic acid components to be assembled into a vector encoding for a
fusion protein comprising a protein or polypeptide covalently operably
linked directly or indirectly to an immobilized metal ion-affinity
peptide. In addition, or alternatively, the kit may be comprised of one
or more of the following: buffers, enzymes, a chromatography column
comprising a resin containing immobilized metal ions and an instructional
brochure explaining how to use the kit.

[0012] Other objects and advantages of the present invention will become
apparent as the detailed description of the invention proceeds.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention generally relates to the expression and
purification of recombinant polypeptides, proteins or protein fragments
containing a metal ion-affinity peptide. In addition to the metal
ion-affinity peptide, the recombinant polypeptides and proteins will
typically also contain a target polypeptide, protein or fragment thereof
covalently linked to the metal ion-affinity peptide. In one embodiment,
the target polypeptide, protein or protein fragment is a biologically
active protein or protein fragment. Advantageously, the metal
ion-affinity peptide enables the recombinant polypeptides and proteins to
be readily purified from a liquid sample by means of metal ion affinity
chromatography.

[0014] The fusion proteins of this invention are prepared by recombinant
DNA methodology. In accordance with the present invention, a gene
sequence coding for a desired protein is isolated, synthesized or
otherwise obtained and operably linked to a DNA sequence coding for the
metal ion-affinity peptide. The hybrid gene containing the gene for a
desired protein operably linked to a DNA sequence encoding the metal
ion-affinity peptide is referred to as a chimeric gene.

[0015] In one embodiment, the metal ion-affinity peptide is covalently
linked to the carboxy terminus of the target polypeptide, protein or
protein fragment. In another embodiment, the metal ion-affinity peptide
is covalently linked to the amino terminus of the target polypeptide,
protein or protein fragment. In each of these embodiments, the metal
ion-affinity peptide and the target polypeptide, protein or protein
fragment may be directly attached by means of a peptide bond or,
alternatively, the two may be separated by a linker. When present, the
linker may provide other functionality to the recombinant polypeptide,
protein or protein fragment.

[0016] The recombinant polypeptides, proteins or protein fragments of the
present invention are defined by the general formula (I):

R1-Sp1-(His-Z1-His-Arg-His-Z2-H is)-Sp2-R2
(I)

wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal
ion-affinity peptide; Z1 is an amino acid residue selected from the
group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro,
Ser, Thr, Trp, and Val; and Z2 is an amino acid residue selected
from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile,
Leu, Lys, Met, Pro, Ser, Thr, Tyr and Val. In addition, R1 is
hydrogen, a polypeptide, protein or protein fragment, Sp1 is a
covalent bond or a spacer comprising at least one amino acid residue,
R2 is hydrogen, a polypeptide, protein or protein fragment, Sp2
is a covalent bond or a spacer comprising at least one amino acid
residue. Thus, for example, R1 or R2 may comprise a target
polypeptide, protein, or protein fragment which is directly or indirectly
linked to the metal ion-affinity peptide.

[0017] Metal Ion-Affinity Peptide

[0018] In one embodiment, the recombinant polypeptide, protein or protein
fragment is defined by formula (I), wherein Z1 is an amino acid
selected from the group consisting of Ala, Asn, Ile, Lys, Phe, Ser, Thr,
and Val; and Z2 is an amino acid selected from the group consisting
of Ala, Asn, Gly, Lys, Ser, Thr, Tyr; and R1, R2, Sp1, and
Sp2 are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R1 or R2)
may be at the carboxy or amino terminus of the metal ion-affinity
polypeptide. In addition, the target polypeptide, protein or protein
fragment (R1 or R2), may be directly fused (when Sp1 or
Sp2 is a covalent bond) or separated from the metal ion-affinity
polypeptide by a spacer (when Sp1 or Sp2 is one or more amino
acid residues) regardless of whether the target polypeptide, protein or
protein fragment is fused to the amino or carboxy terminus of the metal
ion-affinity polypeptide.

[0019] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is an amino
acid selected from the group consisting of Asn and Lys; and Z2 is an
amino acid selected from the group consisting of Gly and Lys; and
R1, R2, Sp1, and Sp2 are as previously defined. For
example, in one such embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I) wherein Z1 is Asn,
Z2 is Lys and R1, R2 Sp1, and Sp2 are as
previously defined. By way of further example, in another such
embodiment, the recombinant polypeptide, protein or protein fragment is
defined by formula (I) wherein Z1 is Lys and Z2 is Gly. In each
of these alternatives, the target polypeptide, protein or protein
fragment (R1 or R2) may be at the carboxy or amino terminus of
the metal ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R1 or R2), may be directly fused
(when Sp1 or Sp2 is a covalent bond) or separated from the
metal ion-affinity polypeptide by a spacer (when Sp1 or Sp2 is
one or more amino acid residues) regardless of whether the target
polypeptide, protein or protein fragment is fused to the amino or carboxy
terminus of the metal ion-affinity polypeptide.

[0020] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is Ile,
Z2 is Asn, and R1, R2, Sp1, and Sp2 are as
previously defined. Thus, for example, in this embodiment the target
polypeptide, protein or protein fragment (R1 or R2) may be at
the carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment (R1 or
R2), may be directly fused (when Sp1 or Sp2 is a covalent
bond) or separated from the metal ion-affinity polypeptide by a spacer
(when Sp1 or Sp2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is fused
to the amino or carboxy terminus of the metal ion-affinity polypeptide.

[0021] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is Thr,
Z2 is Ser, and R1, R2, Sp1, and Sp2 are as
previously defined. Thus, for example, in this embodiment the target
polypeptide, protein or protein fragment (R1 or R2) may be at
the carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment (R1 or
R2), may be directly fused (when Sp1 or Sp2 is a covalent
bond) or separated from the metal ion-affinity polypeptide by a spacer
(when Sp1 or Sp2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is fused
to the amino or carboxy terminus of the metal ion-affinity polypeptide.

[0022] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is Ser,
Z2 is Tyr, and R1, R2, Sp1, and Sp2 are as
previously defined. Thus, for example, in this embodiment the target
polypeptide, protein or protein fragment (R1 or R2) may be at
the carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment (R1 or
R2), may be directly fused (when Sp1 or Sp2 is a covalent
bond) or separated from the metal ion-affinity polypeptide by a spacer
(when Sp1 or Sp2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is fused
to the amino or carboxy terminus of the metal ion-affinity polypeptide.

[0023] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is Val,
Z2 is Ala, and R1, R2, Sp1, and Sp2 are as
previously defined. Thus, for example, in this embodiment the target
polypeptide, protein or protein fragment (R1 or R2) may be at
the carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment (R1 or
R2), may be directly fused (when Sp1 or Sp2 is a covalent
bond) or separated from the metal ion-affinity polypeptide by a spacer
(when Sp1 or Sp2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is fused
to the amino or carboxy terminus of the metal ion-affinity polypeptide.

[0024] In another embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z1 is Ala,
Z2 is Lys, and R1, R2, Sp1, and Sp2 are as
previously defined. Thus, for example, in this embodiment the target
polypeptide, protein or protein fragment (R1 or R2) may be at
the carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment (R1 or
R2), may be directly fused (when Sp1 or Sp2 is a covalent
bond) or separated from the metal ion-affinity polypeptide by a spacer
(when Sp1 or Sp2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is fused
to the amino or carboxy terminus of the metal ion-affinity polypeptide.

[0025] In a further embodiment, R1 may be a polypeptide which drives
expression of the fusion protein and R2 is the target polypeptide,
protein or protein fragment. In this embodiment, each of Sp1 and
Sp2 may be a covalent bond or a spacer, independently of the other.
Thus, for example, R1 may be directly fused to the metal
ion-affinity peptide or separated from the metal ion-affinity peptide by
a spacer independently of whether R2 is directly fused to the metal
ion-affinity peptide or separated from the metal ion-affinity peptide by
a spacer; all of these combinations and permutations are contemplated.
This type of arrangement is particularly useful when chimeric proteins
are constructed which comprise epitopes from two portions of antigenic
protein or from two different antigenic proteins. Such chimeric proteins
may be useful in vaccine preparations.

[0026] In another embodiment, the recombinant polypeptides, proteins or
protein fragments of the present invention comprise multiple copies of
the metal ion-affinity peptide (His-Z1-His-Arg-His-Z2-His) (SEQ
ID NO: 24) wherein Z1 and Z2 are as previously defined. In this
embodiment, the additional copies of the metal affinity peptide may occur
in either or both of the spacer domains (Sp1 and Sp2) or in
either or both of the other domains (R1 and R2) of the
recombinant polypeptides, proteins or protein fragments. Thus, for
example, in one embodiment a second copy of the metal ion-affinity
peptide (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) wherein
Z1 and Z2 are as previously defined is located in one of the
spacer domains (Sp1 or Sp2) or other domains (R1 and
R2) of the recombinant polypeptides, proteins or protein fragments.
By way of further example, in another embodiment two additional copies of
the metal ion-affinity peptide (His-Z1-His-Arg-His-Z2-His) (SEQ
ID NO: 24) wherein Z1 and Z2 are as previously defined are
located in the spacer domains (Sp1 or Sp2) or other domains
(R1 and R2) of the recombinant polypeptides, proteins or
protein fragments. By way of further example, in another embodiment at
least three additional copies of the metal ion-affinity peptide
(His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) wherein Z1 and
Z2 are as previously defined are located in the spacer domains
(Sp1 or Sp2) or other domains (R1 and R2) of the
recombinant polypeptides, proteins or protein fragments. In each of these
embodiments, the multiple copies of the metal ion-affinity peptide may be
separated by one or more amino acid residues (i.e., a spacer) as
described herein. Alternatively, in each of these embodiments the
multiple copies of the metal ion-affinity peptide may be directly linked
to each other without any intervening amino acid residues. Thus, for
example, in one such embodiment the recombinant polypeptides, proteins or
protein fragments of the present invention may be defined by the general
formula (II):

R1-Sp1-(His-Z1-His-Arg-His-Z2-His)t-Sp2-R.-
sub.2 (II)

wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal
ion-affinity peptide; t is at least 2 and R1, R2, Z1,
Z2, Sp1 and Sp2 are as previously defined. By way of
further example, in one such embodiment the recombinant polypeptides,
proteins or protein fragments of the present invention may be defined by
the general formula (III):

R1-Sp1-[(His-Z1-His-Arg-His-Z2-His)-Sp2]t--
R2 (III)

wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal
ion-affinity peptide; t is at least 2 and R1, R2, Z1,
Z2, Sp1 and Sp2 are as previously defined; in addition,
each Sp2 of the recombinant polypeptides, proteins or protein
fragments corresponding to general formula (III) may be the same or
different.

[0027] Target Polypeptide, Protein or Protein Fragment

[0028] The target polypeptide, protein or protein fragment may be composed
of any proteinaceous substance that can be expressed in transformed host
cells. Accordingly, the present invention may be beneficially employed to
produce substantially any prokaryotic or eukaryotic, simple or
conjugated, protein that can be expressed by a vector in a transformed
host cell. For example, the target protein may be [0029] a) an enzyme,
whether oxidoreductase, transferase, hydrolase, lyase, isomerase or
ligase; [0030] b) a storage protein, such as ferritin or ovalbumin or a
transport protein, such as hemoglobin, serum albumin or ceruloplasmin;
[0031] c) a protein that functions in contractile and motile systems such
as actin or myosin; [0032] d) any of a class of proteins that serve a
protective or defense function, such as the blood protein fibrinogen or a
binding protein, such as antibodies or immunoglobulins that bind to and
thus neutralize antigens; [0033] e) a hormone such as human Growth
Hormone, somatostatin, prolactin, estrone, progesterone, melanocyte,
thyrotropin, calcitonin, gonadotropin and insulin; [0034] f) a hormone
involved in the immune system, such as interleukin-1, interleukin-2,
colony stimulating factor, macrophage-activating factor and interferon;
[0035] g) a toxic protein, such as ricin from castor bean or gossypin
from cotton linseed; [0036] h) a protein that serves as structural
elements such as collagen, elastin, alpha-keratin, glyco-proteins, viral
proteins and muco-proteins; or [0037] i) a synthetic protein, defined
generally as any sequence of amino acids not occurring in nature. In
general, the target polypeptide, protein or protein fragment may be a
constituent of the R1 and R2 moieties of the recombinant
polypeptides, proteins or protein fragments corresponding to general
formulae (I), (II) and (III).

[0038] Genes coding for the various types of protein molecules identified
above may be obtained from a variety of prokaryotic or eukaryotic
sources, such as plant or animal cells or bacteria cells. The genes can
be isolated from the chromosome material of these cells or from plasmids
of prokaryotic cells by employing standard, well-known techniques. A
variety of naturally occurring and synthesized plasmids having genes
coding for many different protein molecules are not commercially
available from a variety of sources. The desired DNA also can be produced
from mRNA by using the enzyme reverse transcriptase. This enzyme permits
the synthesis of DNA from an RNA template.

[0039] In one embodiment, R1 may be a protein which enhances
expression and R2 is the target polypeptide, protein, or protein
fragment. It is well known that the presence of some proteins in a cell
result in expression of genes. If a chimeric protein contains an active
portion of the protein which prompts or enhances expression of the gene
encoding it, greater quantities of the protein may be expressed than if
it were not present.

[0040] Linker and Other Optional Elements

[0041] In one embodiment, the recombinant polypeptide, protein or protein
fragment includes a spacer (Sp1 or Sp2) between the metal
ion-affinity polypeptide and the target polypeptide, protein or protein
fragment. If present, the spacer may simply comprise one or more, e.g.,
three to ten amino acid residues, separating the metal ion-affinity
peptide from the target polypeptide, protein or protein fragment.
Alternatively, the spacer may comprise a sequence which imparts other
functionality, such as a proteolytic cleavage site, a fusion protein, a
secretion sequence (e.g. OmpA or OmpT for E. coli, preprotrypsin for
mammalian cells, a-factor for yeast, and melittin for insect cells), a
leader sequence for cellular targeting, antibody epitopes, or IRES
(internal ribosomal entry sequences) sequences.

[0042] In one embodiment, the spacer is selected from among hydrophilic
amino acids to increase the hydrophilic character of the recombinant
polypeptide, protein or protein fragment. Alternatively, the amino
acid(s) of the spacer domain may be selected to impart a desired folding
to the recombinant polypeptide, protein or protein fragment thereby
increasing accessability to one or more regions of the molecule. For
example, the spacer domain may comprise glycine residues which results in
a protein folding conformation which allows for improved accessibility to
antibodies.

[0043] In another embodiment, the spacer comprises a cleavage site which
consists of a unique amino acid sequence cleavable by use of a
sequence-specific proteolytic agent. Such a site would enable the metal
ion-affinity polypeptide to be readily cleaved from the target
polypeptide, protein or protein fragment by digestion with a proteolytic
agent specific for the amino acids of the cleavage site. Alternatively,
the metal ion-affinity peptide may be removed from the desired protein by
chemical cleavage using methods known to the art.

[0044] When present, the cleavable site may be located at the amino or
carboxy terminus of the target peptide. Preferably, the cleavable site is
immediately adjacent the desired protein to enable separation of the
desired protein from the metal ion-affinity peptide. This cleavable site
preferably does not appear in the desired protein. In one embodiment, the
cleavable site is located at the amino terminus of the desired protein.
If the cleavable site is located at the amino terminus of the desired
protein and if there are remaining extraneous amino acids on the desired
protein after cleavage with the proteolytic agent, an endopeptidase such
as trypsin, clostropain or furin may be utilized to remove these
remaining amino acids, thus resulting in a highly purified desired
protein. Further examples of proteolytic enzymatic agents useful for
cleavage are papain, pepsin, plasmin, thrombin, enterokinase, and the
like. Each effects cleavage at a particular amino acid sequence which it
recognizes.

[0045] Digestion with a proteolytic agent may occur while the fusion
protein is still bound to the affinity resin or alternatively, the fusion
protein may be eluted from the affinity resin and then digested with the
proteolytic agent in order to further purify the desired protein.
Preferably, the amino acid sequence of the proteolytic cleavage site is
unique, thus minimizing the possibility that the proteolytic agent will
cleave the desired protein. In one embodiment, the cleavable site
comprises amino acids for an enterokinase, thrombin or a Factor Xa
cleavage site.

[0046] Enterokinase recognizes several sequences: Asp-Lys; Asp-Asp-Lys;
Asp-Asp-Asp-Lys (SEQ ID NO: 25); and Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 26).
The only known natural occurrence of Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 26)
is in the protein trypsinogen which is a natural substrate for bovine
enterokinase and some yeast proteins. As such, by interposing a fragment
containing the amino acid sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 26) as
a cleavable site between the metal ion-affinity polypeptide and the amino
terminus of the target polypeptide, protein or protein fragment, the
metal ion-affinity polypeptide can be liberated from the desired protein
by use of bovine enterokinase with very little likelihood that this
enzyme will cleave any portion of the desired protein itself.

[0047] Thrombin cleaves on the carboxy-terminal side of arginine in the
following sequence: Leu-Val-Pro-Arg-Gly-X (SEQ ID NO: 27), where X is a
non-acidic amino acid. Factor Xa protease (i.e., the activated form of
Factor X) cleaves after the Arg in the following sequences:
Ile-Glu-Gly-Arg-X (SEQ ID NO: 28), Ile-Asp-Gly-Arg-X (SEQ ID NO: 29), and
Ala-Glu-Gly-Arg-X (SEQ ID NO: 30), where X is any amino acid except
proline or arginine. A fusion protein comprising the 31 amino-terminal
residues of the cII protein, a Factor Xa cleavage site and human
β-globin was shown to be cleaved by Factor Xa and generate authentic
β-globin. A limitation of the Factor Xa-based fusion systems is the
fact that Factor Xa has been reported to cleave at arginine residues that
are not present within in the Factor Xa recognition sequence. Lauritzen,
C. et al., Protein Expr. and Purif., 5-6:372-378 (1991).

[0048] While less preferred, other unique amino acid sequences for other
cleavable sites may also be employed in the spacer without departing from
the spirit or scope of the present invention. For instance, the spacer
may be composed, at least in part, of a pair of basic amino acids, i.e.,
Arg, His or Lys. This sequence is cleaved by kallikreins, a glandular
enzyme. Also, the spacer may be composed, at least in part, of Arg-Gly,
since it is known that the enzyme thrombin will cleave after the Arg if
this residue is followed by Gly.

[0049] Regardless of whether a cleavage site is present, the recombinant
polypeptide, protein or protein fragment may comprise an antigenic domain
in a spacer region (Sp1 or Sp2). For example, in one embodiment
of the present invention, the recombinant polypeptide, protein or protein
fragment comprises one or multiple copies of an antigenic domain
generally corresponding to the FLAG® (Sigma-Aldrich, St. Louis, Mo.)
peptide sequence joined to a linking sequence containing a single
enterokinase cleavage site. Such antigenic domains generally correspond
to the sequence:

[0054] each X1 and X4 is independently a covalent bond or at
least one amino acid residue, if other than a covalent bond, preferably
at least one amino acid residue selected from the group consisting of
aromatic amino acid residues and hydrophilic amino acid residues, more
preferably at least one hydrophilic amino acid residue, and still more
preferably at least one an aspartate residue;

[0055] each X2, X3, X7 and X8 is independently an
amino acid residue, preferably an amino acid residue selected from the
group consisting of aromatic amino acid residues and hydrophilic amino
acid residues, more preferably a hydrophilic amino acid residue, and
still more preferably an aspartate residue;

[0056] X5 is a covalent bond or a spacer domain comprising at least
one amino acid, if other than a covalent bond, preferably a histidine
residue, a glycine residue or a combination of multiple or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X)m--, wherein m is 1 to 6 and X is selected from the group
consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;

[0057] X9 is a covalent bond or D; and

[0058] n is 0, 1 or 2.

[0059] In this embodiment, the amino acid sequence
X20--(X1--Y--K--X2--X3-D-X4)n (SEQ ID NO:
35) comprises an antigenic domain --X1--Y--K--X2--X3-D-
(SEQ ID NO: 36) joined in tandem which are joined to a linking sequence
(X1--Y--K--X7--X8-D-X9--K) (SEQ ID NO: 37). The
antigenic domains may be immediately adjacent to each other when n is at
least one and X4 is a covalent bond; optionally, X4 may be a
spacer domain interposed between the multiple copies of antigenic
domains. The linking sequence contains a single enterokinase cleavable
site which is represented by the sequence --X7--X8-D-X9--K
(SEQ ID NO: 38), where X7 and X8 may be an amino acid residue
or a covalent bond and X9 is a covalent bond or an aspartate
residue. In one embodiment, each X7, X8 and X9 is
independently an aspartate residue thus resulting in the enterokinase
cleavable site DDDDK (SEQ ID NO: 26) which is preferably located
immediately adjacent to the amino terminus of the target peptide. When n
is at least one and X5 is a covalent bond, the multiple copies of
antigenic domains may be immediately adjacent to the linking sequence;
optionally, X5 may be a spacer domain interposed between the linking
sequence and the antigenic domains. When each X4 and X5 is
independently a spacer domain, it is preferred that the amino acid
residue(s) of each X4 and X5 impart one or more desired
properties to the antigenic domain; for example, the amino acids of the
spacer domain may be selected to impart a desired folding to the
identification polypeptide thereby increasing accessibility to the
antibody. In another embodiment, the amino acids of the spacer domain
X4 and X5 may be selected to impart a desired affinity
characteristic such as a combination of multiple or alternating histidine
residues capable of chelating to an immobilized metal ion on a resin or
other matrix. Furthermore, these desired properties may be designed into
other areas of the identification polypeptide; for example, the amino
acids represented by X2 and X3 may be selected to impart a
desired peptide folding or a desired affinity characteristic for use in
affinity purification.

[0060] In another embodiment, the spacer comprises multiple copies of an
antigenic domain. For example, in one embodiment the spacer may comprise
a linking sequence containing a single enterokinase or other cleavage
site, or generally correspond to the sequence:

[0064] X20 and X21 are independently a hydrogen or a covalent
bond; each X2, X3, X7 and X8 is independently an
amino acid residue, preferably an amino acid residue selected from the
group consisting of aromatic amino acid residues and hydrophilic amino
acid residues, more preferably a hydrophilic amino acid residue, and
still more preferably an aspartate residue;

[0065] X5 is a covalent bond or a spacer domain comprising at least
one amino acid, if other than a covalent bond, preferably a histidine
residue, a glycine residue or a combination of multiple or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X)m--, wherein m is 1 to 6 and X is selected from the group
consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;

[0066] X9 is a covalent bond or an aspartate residue; and

[0067] n is at least 2.

[0068] In this embodiment, the amino acid sequence
X20-(D-Y--K--X2--X3-D)n (SEQ ID NO: 41) represents
the multiple copies of the antigenic domain D-Y--K--X2--X3-D
(SEQ ID NO: 31) in tandem which are joined to a linking sequence
(D-Y--K--X7--X8-D-X9--K) (SEQ ID NO: 32). In this
embodiment, one antigenic domain is immediately adjacent to another
antigenic domain, i.e., no intervening spacer domains, and the multiple
copies of the antigenic domain are immediately adjacent to the linking
sequence when X5 is a covalent bond. The linking sequence contains a
single enterokinase cleavable site which is represented by the sequence
--X7--X8-D-X9-K (SEQ ID NO: 38), where X7 and X8
may be a covalent bond or an amino acid residue, preferably an aspartate
residue, and X9 is a covalent bond or an aspartate residue. In one
embodiment, each X7, X8 and X9 is independently an
aspartate residue thus resulting in the enterokinase cleavable site DDDDK
(SEQ ID NO: 26) which is preferably adjacent to the amino terminus of the
target peptide. Optionally, the multiple copies of the antigenic domain
are joined to the linking sequence by a spacer X5 when X5 is at
least one amino acid residue. When X5 is a spacer domain, it is
preferred that the amino acid residue(s) of X5 impart one or more
desired properties to the recombinant polypeptide, protein or protein
fragment; for example, the amino acids of the spacer domain may be
selected to impart a desired folding to the recombinant polypeptide,
protein or protein fragment thereby increasing accessibility to the
antibody. In another embodiment, the amino acids of the spacer domain may
be selected to impart a desired affinity characteristic such as a
combination of multiple or alternating histidine residues capable of
chelating to an immobilized metal ion on a resin or other matrix.
Furthermore, these desired properties may be designed into other areas of
the spacer; for example, the amino acids represented by X2 and
X3 may be selected to impart a desired peptide folding or a desired
affinity characteristic for use in affinity purification.

[0069] When the affinity polypeptide is located at the amino terminus of
the target polypeptide, protein or protein fragment, it is often
desirable to design the amino acid sequence such that an initiator
methionine is present. Accordingly, in one embodiment of the present
invention, the recombinant polypeptide, protein or protein fragment
comprises multiple copies of an antigenic domain, a linking sequence
containing a single enterokinase cleavage site and generally corresponds
to the sequence:

[0074] X10 is a covalent bond or an amino acid, if other than a
covalent bond, preferably a methionine residue;

[0075] each X2, X3, X7 and X5 is independently an
amino acid residue, preferably an amino acid residue selected from the
group consisting of aromatic amino acid residues and hydrophilic amino
acid residues, more preferably a hydrophilic amino acid residue, and
still more preferably an aspartate residue;

[0076] X5 is a covalent bond or a spacer domain comprising at least
one amino acid, if other than a bond, preferably a histidine residue, a
glycine residue or a combination of multiple or alternating histidine
residues, said combination comprising His-Gly-His, or -(His-X)m--,
wherein m is 1 to 6 and X is selected from the group consisting of Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, Tyr, and Val;

[0077] X9 is a covalent bond or an aspartate residue; and

[0078] n is at least 2.

[0079] In this embodiment, the amino acid sequence
X20--X10-(D-Y--K--X2--X3-D)n (SEQ ID NO: 44)
represents the multiple copies of the antigenic domain
D-Y--K--X2--X3-D (SEQ ID NO: 31) in tandem which is flanked by
a linking sequence (D-Y--K--X7--X8-D-X9--K) (SEQ ID NO:
32) and an initiator amino acid X10, preferably methionine. The
antigenic domain D-Y--K--X2--X3-D with an initiator methionine
is recognized by the M5® antibody (Sigma-Aldrich, St. Louis, Mo.). In
this embodiment, one antigenic domain is immediately adjacent to another
antigenic domain, i.e., no intervening spacer domains, and the multiple
copies of the antigenic domain are immediately adjacent to the linking
sequence when X5 is a covalent bond. The linking sequence contains
an enterokinase cleavable site which is represented by the amino acid
sequence --X7--X8-D-X9--K (SEQ ID NO: 38), where X7
and X8 may be a covalent bond or an amino acid residue, preferably
an aspartate residue, and X9 is a covalent bond or an aspartate
residue. In one embodiment, each X7, X8 and X9 is
independently an aspartate residue thus resulting in the enterokinase
cleavable site DDDDK (SEQ ID NO: 26) which is preferably adjacent to the
amino terminus of the target peptide. Optionally, the multiple copies of
the antigenic domain are joined to the linking sequence by a spacer
domain X5 whenX5 is at least one amino acid residue. When
X5 is a spacer domain, it is preferred that the amino acid
residue(s) of X5 impart one or more desired properties to the
affinity polypeptide; for example, the amino acids of the spacer domain
may be selected to impart a desired folding to the recombinant
polypeptide, protein or protein fragment thereby increasing accessibility
to the antibody. In another embodiment, the amino acids of the spacer
domain may be selected to impart a desired affinity characteristic such
as a combination of multiple or alternating histidine residues capable of
chelating to an immobilized metal ion on a resin or other matrix.
Furthermore, these desired properties may be designed into other areas of
the affinity polypeptide; for example, the amino acids represented by
X2 and X3 may be selected to impart a desired peptide folding
or a desired affinity characteristic for use in affinity purification.

[0080] In another embodiment of the present invention, the recombinant
polypeptide, protein or protein fragment comprises one or more copies of
an antigenic sequence, a linking sequence containing a single
enterokinase cleavable site and generally corresponds to the sequence:

[0086] each X12 is an amino acid, preferably selected from the group
consisting of aromatic amino acid residues and hydrophilic amino acid
residues, more preferably a hydrophilic amino acid residue, and still
more preferably an aspartate residue;

[0087] each X13 is a covalent bond or at least one amino acid, if
other than a covalent bond, preferably selected from the group consisting
of aromatic amino acid residues and hydrophilic amino acid residues, more
preferably a hydrophilic amino acid residue, and still more preferably an
aspartate residue;

[0088] X14 is a covalent bond or a spacer domain comprising at least
one amino acid, if other than a covalent bond, preferably a histidine
residue, a glycine residue or a combination of multiple or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X)m--, wherein m is 1 to 6 and X is selected from the group
consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;

[0089] X15 is a covalent bond or an aspartate residue; and

[0090] n is 0 or at least 1.

[0091] In this embodiment, when n is at least 2, the amino acid sequence
X20-(D-X11--Y--X12--X13)n (SEQ ID NO: 43)
constitutes multiple copies of the antigenic domain
D-X11--Y--X12--X13 (SEQ ID NO: 33) in tandem which are
joined to a linking sequence
(D-X11--Y--X12--X13-D-X15--K) (SEQ ID NO: 34).
Additionally, one antigenic domain may be immediately adjacent to another
antigenic domain, i.e., no intervening spacer domains, and the multiple
copies of the antigenic domain may be immediately adjacent to the linking
sequence when X14 is a covalent bond. The linking sequence contains
a single enterokinase cleavable site which is represented by the sequence
--X12--X13-D-X15--K, (SEQ ID NO: 38) where X12 and
X13 may be a covalent bond or an amino acid residue, preferably an
aspartate residue, and X15 is a covalent bond or an aspartate
residue. In one embodiment, each X12, X13 and X15 is
independently an aspartate residue thus resulting in the enterokinase
cleavable site DDDDK (SEQ ID NO: 26) which is preferably adjacent to the
amino terminus of the target peptide. Optionally, when n is at least two,
the multiple copies of the antigenic domain are joined to the linking
sequence by a spacer X14 when X14 is at least one amino acid
residue. When X14 is a spacer domain, it is preferred that the amino
acid residue(s) of X14 impart one or more desired properties to the
recombinant polypeptide, protein or protein fragment; for example, the
amino acids of the spacer domain may be selected to impart a desired
folding to the recombinant polypeptide, protein or protein fragment
thereby increasing accessibility to the antibody. In another embodiment,
the amino acids of the spacer domain X14 may be selected to impart a
desired affinity characteristic such as a combination of multiple or
alternating histidine residues capable of chelating to an immobilized
metal ion on a resin or other matrix.

[0092] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the enzyme glutathione-S-transferase of the parasite
helminth Schistosoma japonicum (SEQ ID NO: 1). The
glutathione-S-transferase may, however, be derived from other species
including human and other mammalian glutathione-S-transferase. Proteins
expressed as fusions with the enzyme glutathione-S-transferase can be
purified under non-denaturing conditions by affinity chromatography on
immobilized glutathione. Glutathione-agarose beads have a capacity of at
least 8 mg fusion protein/ml swollen beads and can be used several times
for different preparations of the same fusion protein. Smith, D. B. and
Johnson, K. S., Gene, 67:31-40, 1988.

[0093] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises a cellulose binding domain (CBD) (SEQ ID NO: 2).
CBD's are found in both bacterial and fungal sources and possess a high
affinity for the crystalline form of cellulose. This property has been
useful for purification of fusion proteins using a cellulose matrix.
Fusion proteins have been attached at both the N- and C-terminus of CBD.

[0094] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the Maltose Binding Protein (MBP) encoded by the malE
gene in E. coli (SEQ ID NO: 3). MBP has found utility in the formation of
chimeric proteins with eukaryotic proteins for expression in bacterial
systems. This system permits expression of soluble fusion proteins that
can readily be purified on immobilized amylose resin.

[0095] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises Protein A (SEQ ID NO: 4). Protein A is isolated from
Staphylococcus aureus and binds to the Fc origin of IgG. Fusion proteins
containing the IgG binding domains of Protein A can be affinity purified
on IgG resins (e.g., IgG Sepharose 6FF (Pharmacia Biotech). The signal
sequence of Protein A is functional in E. coli. Fusion proteins using
Protein A have shown increased stability when expressed both in the
cytoplasm and periplasm in E. coli.

[0096] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises Protein G (SEQ ID NO: 5). Protein G is similar to
Protein A with the difference being that Protein G binds to human serum
albumin in addition to IgG. The major disadvantage is that low pH<3.4
is required to elute the fusion protein.

[0097] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises IgG (SEQ ID NO: 6). Placing the protein of interest
on the C-terminal of IgG generates chimeric proteins. This allows
purification of the fusion protein using either Protein A or G matrix.

[0098] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the enzyme chloramphenicol acetyl transferase (CAT)
from E. coli (SEQ ID NO: 7). CAT is used in the form of a C-terminal
fusion. CAT is readily translated in E. coli and allows for
over-expression of heterologous proteins. Capture of fusion proteins is
accomplished using a chloramphenicol matrix.

[0099] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises streptavidin (SEQ ID NO: 8). Streptavidin is used for
fusion proteins because of its high affinity and high specificity for
biotin. Streptavidin is a neutral protein, free from carbohydrates and
sulphydryl groups.

[0100] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises b-galactosidase (SEQ ID NO: 9). b-galactosidase is a
enzyme that is utilized as both an N- and C-terminal fusion protein.
Fusion proteins containing b-galactosidase sequences can be affinity
purified on
aminophenyl-b-D-thiogalactosidyl-succinyldiaminohexyl-Sepharose. However,
given that C-terminal fusion proteins are usually insoluble, the system
has limited use in bacterial systems. N-terminal fusions are soluble in
E. coli, but due to the large size of b-galactosidase, this system is
used more often in eukaryotic gene expression.

[0101] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the Green Fluorescent Protein (GFP) (SEQ ID NO: 10).
GFP is a protein from the jellyfish Aquorea victorea and many mutant
variations of this protein have been used successfully in most organisms
for protein expression. The major use of these types of fusion proteins
is for targeting and determining physiological function of the host cell
protein.

[0102] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises thioredoxin (SEQ ID NO: 11). Thioredoxin is a
relatively small thermostable protein that is easily over-expressed in
bacterial systems. Thioredoxin fusion systems are useful in avoiding the
formation of inclusion bodies during heterologous gene expression. This
has been particularly useful in the expression of mammalian cytokines.

[0103] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises Calmodulin Binding Protein (CBP) (SEQ ID NO: 12).
This tag is derived from the C-terminus of skeletal muscle myosin light
chain kinase. This small tag is recognized by calmodulin and forms the
base of the technology. The tag is translated efficiently and allows for
the expression and recovery of N-terminal chimeric genes.

[0104] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the c-myc epitope sequence
Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 13). This C-terminal
portion of the myc oncogene, which is part of the p53 signaling pathway,
has been used as a detection tag for expression of recombinant proteins
in mammalian cells.

[0105] In another embodiment of this invention, a spacer (Sp1 or
Sp2) comprises the HA epitope sequence Tyr-Pro-Tyr-Asp-Val-Tyr-Ala
(SEQ ID NO: 14). This detection tag has been utilized for the expression
of recombinant proteins in mammalian cells.

[0106] In another embodiment of this invention, the spacer (Sp1 or
Sp2) comprises a polypeptide possessing an amino acid sequence
having at least 70% homology to any one of the amino acid sequences
disclosed in SEQ ID NOS:1-14, and retains the same binding
characteristics as said amino acid sequence.

[0107] DNA sequences encoding the aforementioned proteins which may be
employed as spacers (Sp1 or Sp2) are commercially available
(e.g., malE gene sequences encoding the MBP are available from New
England Biolabs (pMAL-c2 and pMAL-p2); Schistosoma japonicum
glutathione-S-transferase (GST) gene sequences are available from
Pharmacia Biotech (the pGEX series which have GenBank Accession Nos.:
U13849 to U13858); β-galactosidase (the lacZ gene product) gene
sequences are available from Pharmacia Biotech (pCH110 and pMC1871;
GenBank Accession Nos: U13845 and L08936, respectively); sequences
encoding the IgG binding domains of Protein A are available from
Pharmacia Biotech (pRIT2T; GenBank Accession No. U13864)).

[0108] When any of the above listed proteins (including the hinge/Fc
domains of human IgG1) are used as spacers, it is not required that
the entire protein be used as a spacer. Portions of these proteins may be
used as the spacer provided the portion selected is sufficient to permit
interaction of a fusion protein containing the portion of the protein
used as the spacer with the desired affinity resin.

[0109] Expression and Purification

[0110] The polypeptides, proteins and protein fragments of the present
invention are generally prepared and expressed as a fusion protein using
conventional recombinant DNA technology. The fusion protein is thus
produced by host cells transformed with the genetic information encoding
the fusion protein. The host cells may secrete the fusion protein into
the culture media or store it in the cells whereby the cells must be
collected and disrupted in order to extract the product. As hosts, E.
coli, yeast, insect cells, mammalian cells and plants are suitable. Of
these two, E. coli will typically be the more preferred host for most
applications. In one embodiment, the recombinant polypeptides, proteins
and protein fragments are produced in a soluble form or secreted from the
host.

[0111] In general, a chimeric gene is inserted into an expression vector
which allows for the expression of the desired fusion protein in a
suitable transformed host. The expression vector provides the inserted
chimeric gene with the necessary regulatory sequences to control
expression in the suitable transformed host.

[0112] There are six elements of control expression sequence for proteins
which are to be secreted from a host into the medium, while five of these
elements apply to fusion proteins expressed intracellularly. These
elements in the order they appear in the gene are: a) the promoter
region; b) the 5' untranslated region; c) signal sequence; d) the
chimeric coding sequence; e) the 3' untranslated region; f) the
transcription termination site. Fusion proteins which are not secreted do
not contain c), the signal sequence.

[0113] The recombinant expression vectors of the invention comprise a
nucleic acid of the invention in a form suitable for expression of the
nucleic acid in a host cell. This means that the recombinant expression
vectors include one or more regulatory sequences, selected on the basis
of the host cells to be used for expression, operably linked to the
nucleic acid sequence to be expressed. It will be appreciated by those
skilled in the art that the design of the expression vector can depend on
such factors as the choice of the host cell to be transformed, the level
of expression of protein desired, etc. The expression vectors of the
invention can be introduced into host cells to thereby produce proteins
or peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein.

[0114] Vector DNA can be introduced into prokaryotic or eukaryotic cells
via conventional transformation or transfection techniques. For stable
transfection of mammalian cells, it is known that, depending upon the
expression vector and transfection technique used, only a small fraction
of cells may integrate the foreign DNA into their genome. In order to
identify and select these integrants, a gene that encodes a selectable
marker (e.g., for resistance to antibiotics) is generally introduced into
the host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as G418,
hygromycin, and methotrexate. Nucleic acid encoding a selectable marker
can be introduced into a host cell on the same vector as that encoding
the metal ion-affinity peptide containing fusion protein or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g., cells
that have incorporated the selectable marker gene will survive, while the
other cells die). Methods and materials for preparing recombinant
vectors, transforming host cells using replicating vectors, and
expressing biologically active foreign polypeptides and proteins are
generally well known.

[0115] The expressed recombinant polypeptides, proteins and protein
fragments may be separated from other material present in the secretion
media or extraction solution, or from other liquid mixtures, through
immobilized metal affinity chromatography ("IMAC"). For example, the
culture media containing the secreted recombinant polypeptides, proteins
and protein fragments or the cell extracts containing the recombinant
polypeptides, proteins and protein fragments may be passed through a
column that contains a resin comprising an immobilized metal ion. In
IMAC, metal ions are immobilized onto to a solid support, and used to
capture proteins comprising a metal chelating peptide. The metal
chelating peptide may occur naturally in the protein, or the protein may
be a recombinant protein with an affinity tag comprising a metal
chelating peptide. Exemplary metal ions include aluminum, cadmium,
calcium, cobalt, copper, gallium, iron, nickel, ytterbium and zinc. In
one embodiment, the metal ion is preferably nickel, copper, cobalt, or
zinc. In another embodiment, the metal ion is nickel. Advantageously, the
components of the solution other than recombinant polypeptide, protein or
protein fragment freely pass through the column. The immobilized metal,
however, chelates or binds the recombinant polypeptides, proteins and
protein fragments, thereby separating it from the remaining contents of
the liquid mixture in which it was originally contained.

[0117] In one embodiment, the capture ligand is a metal chelate as
described in WO 01/81365. More specifically, in this embodiment the
capture ligand is a metal chelate derived from metal chelating
composition (1):

##STR00001##

wherein [0118] Q is a carrier; [0119] S1 is a spacer; [0120] L is
-A-T-CH(X)-- or --C(═O)--; [0121] A is an ether, thioether,
selenoether, or amide linkage; [0122] T is a bond or substituted or
unsubstituted alkyl or alkenyl; [0123] X is --(CH2)kCH3,
--(CH2)kCOOH,--(CH2)kSO3H,
--(CH2)kPO3H2, --(CH2)kN(J)2, or
--(CH2)kP(J)2, preferably --(CH2)kCOOH or
--(CH2)kSO3H; [0124] k is an integer from 0 to 2; [0125] J
is hydrocarbyl or substituted hydrocarbyl; [0126] Y is --COOH, --H,
--SO3H, --PO3H2, --N(J)2, or --P(J)2,
preferably, --COOH; [0127] Z is --COOH, --H, --SO3H,
--PO3H2, --N(J)2, or --P(J)2, preferably, --COOH; and
[0128] i is an integer from 0 to 4, preferably 1 or 2.

[0129] In general, the carrier, Q, may comprise any solid or soluble
material or compound capable of being derivatized for coupling. Solid (or
insoluble) carriers may be selected from a group including agarose,
cellulose, methacrylate co-polymers, polystyrene, polypropylene, paper,
polyamide, polyacrylonitrile, polyvinylidene, polysulfone,
nitrocellulose, polyester, polyethylene, silica, glass, latex, plastic,
gold, iron oxide and polyacrylamide, but may be any insoluble or solid
compound able to be derivatized to allow coupling of the remainder of the
composition to the carrier, Q. Soluble carriers include proteins, nucleic
acids including DNA, RNA, and oligonucleotides, lipids, liposomes,
synthetic soluble polymers, proteins, polyamino acids, albumin,
antibodies, enzymes, streptavidin, peptides, hormones, chromogenic dyes,
fluorescent dyes, flurochromes or any other detection molecule, drugs,
small organic compounds, polysaccharides and any other soluble compound
able to be derivatized for coupling the remainder of the composition to
the carrier, Q. In one embodiment, the carrier, Q, is the container of
the present invention. In another embodiment, the carrier, Q, is a body
provided within the container of the present invention.

[0130] The spacer, S1, which flanks the carrier comprises a chain of
atoms which may be saturated or unsaturated, substituted or
unsubstituted, linear or cyclic, or straight or branched. Typically, the
chain of atoms defining the spacer, S1, will consist of no more than
about 25 atoms; stated another way, the backbone of the spacer will
consist of no more than about 25 atoms. More preferably, the chain of
atoms defining the spacer, S1, will consist of no more than about 15
atoms, and still more preferably no more than about 12 atoms. The chain
of atoms defining the spacer, S1, will typically be selected from
the group consisting of carbon, oxygen, nitrogen, sulfur, selenium,
silicon and phosphorous and preferably from the group consisting of
carbon, oxygen, nitrogen, sulfur and selenium. In addition, the chain
atoms may be substituted or unsubstituted with atoms other than hydrogen
such as hydroxy, keto (═O), or acyl such as acetyl. Thus, the chain
may optionally include one or more ether, thioether, selenoether, amide,
or amine linkages between hydrocarbyl or substituted hydrocarbyl regions.
Exemplary spacers, S1, include methylene, alkyleneoxy
(--(CH2)aO--), alkylenethioether (--(CH2)aS--),
alkyleneselenoether (--(CH2)aSe--), alkyleneamide
(--(CH2)aNR1(C═O)--), alkylenecarbonyl
(--(CH2)aCO)--, and combinations thereof wherein a is generally
from 1 to about 20 and R1 is hydrogen or hydrocarbyl, preferably
alkyl. In one embodiment, the spacer, S1, is a hydrophilic, neutral
structure and does not contain any amine linkages or substituents or
other linkages or substituents which could become electrically charged
during the purification of a polypeptide.

[0131] As noted above, the linker, L, may be -A-T-CH(X)-- or
--C(═O)--. When L is -A-T-CH(X)--, the chelating composition
corresponds to the formula:

##STR00002##

wherein Q, S1, A, T, X, Y, and Z are as previously defined. In this
embodiment, the ether (--O--), thioether (--S--), selenoether (--Se--) or
amide ((--NR1(C═O)--) or (--(C═O)NR1--) wherein R1
is hydrogen or hydrocarbyl) linkage is separated from the chelating
portion of the molecule by a substituted or unsubstituted alkyl or
alkenyl region. If other than a bond, T is preferably substituted or
unsubstituted C1 to C6 alkyl or substituted or unsubstituted
C2 to C6 alkenyl. More preferably, A is --S--, T is
--(CH2)n--, and n is an integer from 0 to 6, typically 0 to 4,
and more typically 0, 1 or 2.

[0132] When L is --C(═O)--, the chelating composition corresponds to
the formula:

##STR00003##

wherein Q, S1, i, Y, and Z are as previously defined.

[0133] In one embodiment, the sequence --S1-L-, in combination, is a
chain of no more than about 35 atoms selected from the group consisting
of carbon, oxygen, sulfur, selenium, nitrogen, silicon and phosphorous,
more preferably only carbon, oxygen sulfur and nitrogen, and still more
preferably only carbon, oxygen and sulfur. To reduce the prospects for
non-specific binding, nitrogen, when present, is preferably in the form
of an amide moiety. In addition, if the carbon chain atoms are
substituted with anything other than hydrogen, they are preferably
substituted with hydroxy or keto. In one embodiment, L comprises a
portion (sometimes referred to as a fragment or residue) derived from an
amino acid such as cystine, homocystine, cysteine, homocysteine, aspartic
acid, cysteic acid or an ester thereof such as the methyl or ethyl ester
thereof.

[0135] In another embodiment, the capture ligand is a metal chelate of the
type described in U.S. Pat. No. 5,047,513. More specifically, in this
embodiment the capture ligand is a metal chelate derived from
nitrilotriacetic acid derivatives of the formula

##STR00009##

wherein S2 is --O--CH2--CH(OH)--CH2 or --O--CO-- and x is
2, 3 or 4. In this embodiment, the nitrilotriacetic acid derivative is
immobilized on any of the previously described carriers, Q.

[0136] In these embodiments in which the capture ligand is a metal chelate
as described in WO 01/81365 or U.S. Pat. No. 5,047,513, the metal chelate
may contain any of the metal ions previously described in connection with
IMAC. In one embodiment, the metal chelate comprises a metal ion selected
from among nickel (Ni2+), zinc (Zn2+), copper (Cu2+), iron
(Fe3+), cobalt (Co2+), calcium (Ca2+), aluminum
(Al3+), magnesium (Mg2+), and manganese (Mn2+). In another
embodiment, the metal chelate comprises nickel (Ni2+).

[0137] Another common purification technique that can be used in the
context of the present invention is the use of an immunogenic capture
system where the recombinant polypeptide, protein or protein fragment
comprises an antigenic domain in a spacer region (Sp1 or Sp2).
Any of the previously described antigenic systems comprising the spacer
may be used for this purpose. In such systems, an epitope tag on a
protein or peptide allows the protein to which it is attached to be
purified based upon the affinity of the epitope tag for a corresponding
ligand (e.g., antibody) immobilized on a support. One example of such a
tag is the sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 15), or
DYKDDDDK (SEQ ID NO: 15); antibodies having specificity for this sequence
are sold by Sigma-Aldrich (St. Louis, Mo.) under the FLAG® trademark.
Another example of such a tag is the sequence
Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 16), or DLYDDDDK (SEQ ID NO:
16); antibodies having specificity for this sequence are sold by
Invitrogen (Carlsbad, Calif.). Another example of such a tag is the
3× FLAG® sequence
Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-A-
sp-Asp-Asp-Asp-Lys (SEQ ID NO: 17); antibodies having specificity for this
sequence are sold by Sigma-Aldrich (St. Louis, Mo.). Thus, in one
embodiment, the carrier comprises immobilized antibodies which have
specificity for the DYKDDDDK (SEQ ID NO: 15) epitope; in another
embodiment, the carrier comprises immobilized antibodies which have
specificity for the DLYDDDDK (SEQ ID NO: 16) epitope. In another
embodiment, the carrier comprises immobilized antibodies which have
specificity for SEQ ID NO: 17. For example, in one embodiment, the
ANTI-FLAG® M1, M2, or M5 antibody is immobilized on the interior
surface of a column, or a portion thereof, and/or a bead or other support
within a column.

[0138] After the recombinant polypeptides, proteins and protein fragments
are separated from other components of the liquid mixture, the conditions
in the column may be changed to release the bound material. For example,
the bound molecules may be eluted by pH change, imidazole, or competition
with another linker peptide from the column.

[0139] Alternatively, the target polypeptide, protein or protein fragment
portion of the bound recombinant polypeptide, protein or protein fragment
may be selectively released from immobilized metal. For example, if there
is a cleavage site between the target polypeptide, protein or protein
fragment and the metal ion-affinity peptide, and if the bound recombinant
polypeptide, protein or protein fragment is treated with the appropriate
enzyme, the target polypeptide, protein or protein fragment may be
selectively released while the metal ion-affinity polypeptide fragment
remains bound to the immobilized metal. For this purpose, the cleavage is
preferably an enzymatically cleavable linker peptide having the ability
to undergo site-specific proteolysis. Suitable cleaving enzymes in
accordance with this invention are activated factor X (factor Xa), DPP I,
DPP II, DPP IV, carboxylpeptidase A, collagen, enterokinase, human renin,
thrombin, trypsin, ubtilisn and V5.

[0140] It is to be appreciated that some polypeptide or protein molecules
will possess the desired enzymatic or biological activity with the metal
chelate peptide still attached either at the C-terminal end or at the
N-terminal end or both. In those cases the purification of the chimeric
protein will be accomplished without subjecting the protein to
site-specific proteolysis.

[0141] The present invention may be used to purify any prokaryotic or
eukaryotic protein that can be expressed as the product of recombinant
DNA technology in a transformed host cell. These recombinant protein
products include hormones, receptors, enzymes, storage proteins, blood
proteins, mutant proteins produced by protein engineering techniques, or
synthetic proteins. The purification process of the present invention can
be used batchwise or in continuously run columns.

[0142] It is to be understood that the present invention has been
described in detail by way of illustration and example in order to
acquaint others skilled in the art with the invention, its principles,
and its practical application. Further, the specific embodiments of the
present invention as set forth are not intended to be exhaustive or to
limit the invention, and that many alternatives, modifications, and
variations will be apparent to those skilled in the art in light of the
foregoing examples and detailed description. Accordingly, this invention
is intended to embrace all such alternatives, modifications, and
variations that fall within the spirit and scope of the following claims.
While some of the examples and descriptions above include some
conclusions about the way the invention may function, the inventors do
not intend to be bound by those conclusions and functions, but put them
forth only as possible explanations in light of current understanding.

Abbreviations and Definitions

[0143] To facilitate understanding of the invention, a number of terms are
defined below. Definitions of certain terms are included here. Any term
not defined is understood to have the normal meaning used by scientists
contemporaneous with the submission of this application.

[0144] The term "expression vector" as used herein refers to nucleic acid
sequences containing a desired coding sequence and appropriate nucleic
acid sequences necessary for the expression of the operably linked coding
sequence in a particular host organism. Nucleic acid sequences necessary
for expression in prokaryotes include a promoter, a ribosome binding
site, an initiation codon, a stop codon, optionally an operator sequence
and possibly other regulatory sequences. Eukaryotic cells utilize
promoters, a Kozak sequence and often enhancers and polyadenlyation
signals. Prokaryotic cells also utilize a Shine-Dalgarno Ribosome binding
site. The present invention includes vectors or plasmids which can be
used as vehicles to transform any viable host cell with the recombinant
DNA expression vector.

[0145] "Operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner that
allows for expression of the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when the vector is
introduced into the host cell).

[0146] The term "regulatory sequence" is intended to include promoters,
enhancers, and other expression control elements (e.g., polyadenylation
signals). Regulatory sequences include those that direct constitutive
expression of a nucleotide sequence in many types of host cell and those
that direct expression of the nucleotide sequence only in certain host
cells (e.g., tissue-specific regulatory sequences).

[0147] The terms "transformation" and "transfection" are intended to refer
to a variety of art-recognized techniques for introducing foreign nucleic
acid (e.g., DNA) into a host cell, including calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, or electroporation. Suitable methods for transforming or
transfecting host cells can be found in laboratory manuals.

[0149] The term "hydrophobic" when used in reference to amino acids refers
to those amino acids which have nonpolar side chains. Hydrophobic amino
acids include valine, leucine, isoleucine, cysteine and methionine. Three
hydrophobic amino acids have aromatic side chains. Accordingly, the term
"aromatic" when used in reference to amino acids refers to the three
aromatic hydrophobic amino acids phenylalanine, tyrosine and tryptophan.

[0150] The term "fusion protein" refers to polypeptides and proteins which
consist of a metal ion-affinity linker peptide and a protein or
polypeptide operably linked directly or indirectly to the metal
ion-affinity peptide. The metal ion-affinity linker peptide may be
located at the amino-terminal portion of the fusion protein or at the
carboxy-terminal protein thus forming an "amino-terminal fusion protein"
or a "carboxy-terminal fusion protein," respectively.

[0151] The terms "metal ion-affinity peptide", "metal binding peptide" and
"linker peptide" are used interchangeably to refer to an amino acid
sequence which displays an affinity to metal ions. The minimum length of
the immobilized metal ion-affinity peptide according to the present
invention is seven amino acids including four alternating histidines. The
most preferred length is seven amino acids including four alternating
histidines.

[0152] The term "enzyme" referred to herein in the context of a cleavage
enzyme means a polypeptide or protein which recognizes a specific amino
acid sequence in a polypeptide and cleaves the polypeptide at the
scissile bond. In one embodiment of the present invention, enterokinase
is the enzyme which is used to free the fusion protein from the
immobilized metal ion column. In further embodiments, carboxylpeptidase
A, DPP I, DPP II, DPP IV, factor Xa, human renin, TEV, thrombin or VIII
protease is the enzyme.

[0153] The terms "cleavage site" used herein refers to an amino acid
sequence which is recognized and cleaved by an enzyme or chemical means
at the scissile bond.

[0154] The term "scissile bond" referred to herein is the juncture where
cleavage occurs; for example the scissile bond recognized by enterokinase
may be the bond following the sequence (Asp4)-Lys in the spacer
peptide or affinity peptide.

[0155] By the term "immobilized metal ion-affinity peptide" as used herein
is meant an amino acid sequence that chelates immobilized divalent metal
ions of metals selected from the group consisting of aluminum, cadmium,
calcium, cobalt, copper, gallium, iron, nickel, ytterbium and zinc.

[0156] The term "capture ligand" means any ligand or receptor that can be
immobilized or supported on a container or support and used to isolate a
cellular component from cellular debris. Some non-limiting examples of
capture ligands that may be used in connection with the present invention
include: biotin, streptavidin, various metal chelate ions, antibodies,
various charged particles such as those for use in ion exchange
chromatography, various affinity chromatography supports, and various
hydrophobic groups for use in hydrophobic chromatography.

[0157] For all the nucleotide and amino acid sequences disclosed herein,
it is understood that equivalent nucleotides and amino acids can be
substituted into the sequences without affecting the function of the
sequences. Such substitutions are within the ability of a person of
ordinary skill in the art.

[0158] The procedures disclosed herein which involve the molecular
manipulation of nucleic acids are known to those skilled in the art.

EXAMPLES

Example 1

Construction and Screening of a Metal Ion-Affinity Peptide Library

[0159] A pseudo-random glutathione-S-transferase C-terminal peptide
library was constructed with the amino acid sequence of
His-X-His-X-His-X-His where X is any amino acid except Gln, His and Pro.
The library vector was constructed from the bacterial expression vector
pGEX-2T. The library was constructed by annealing a pair of complimentary
oligonucleotides together. Oligonucleotides were constructed as follows:
5'GATCCCATDNDCATDNDCATDNDCATTAAC3' (SEQ ID NO: 18) and
5'AATTGTTAATGHNHATGHNHATGHNHATGG3' (SEQ ID NO: 19) where D is nucleotides
A, G, or T, H is nucleotides A, C, or T and N is nucleotides A, C, T, or
G. The 5' end was phosphorylated with T4 polynucleotide kinase and
the oligonucleotides were annealed together to generate a cassette. The
cassette was ligated into pGEX-2T, which had been digested with EcoRI and
BamHI restriction endonucleases. Ligated vector was transformed into E.
coli DH5-α using standard protocols. Transformants were plated on
LB/ampicillin plates (100 mg/L) and incubated overnight at 37° C.

[0166] The filters were dried at ambient temperature followed by an
incubation in Tris-buffered saline (TBS) containing 3% non-fat dry milk
for 1 hour at room temperature. Filters were then washed 3× for 5
minutes with TBS containing 0.05% Tween-20 (TBS-T). To detect clones that
were capable of binding to a metal ion, the filters were incubated with
nickel NTA horseradish peroxidase (HRP) at a concentration of 1 mg/ml in
TBS-T for 1 hour. The filters were then washed with TBS-T 3× for 5
minutes and incubated with 3-3'-5-5'-Tetramethylbenzidine (TMB) to detect
the horseradish peroxidase. The reaction was stopped by placing the
filters in water. 250 colonies, which were detected above, were picked
from the master plate and placed into 1 ml of LB/ampicillin and grown
overnight in a 96 deep well plate at 37° C. at 250 rpm on an
orbital shaker. 10 μl of the overnight cultures were transferred to a
fresh aliquot of LB/ampicillin (1 ml) in a 96 deep well plate and grown
for an additional 3 hours. The culture was then induced by adding IPTG
(final concentration of 1 mM) and the culture was allowed to grow for an
additional 3 hours prior to harvesting by centrifugation. The media was
decanted and the cells were frozen overnight at -20° C. in the
collection plate. Cells were lysed with 0.6 ml of CelLytic-B
(Sigma-Aldrich product no. B3553) and incubated for 15 minutes at room
temperature. The cell debris was removed by centrifugation at
3,000×g for 15 minutes. Two experiments were done in parallel, one
on a His-Select High Sensitivity (HS) nickel coated plate, and the second
on HIS-Select High Capacity (HC) nickel coated plate. 0.1 ml of cell
extracts of each clone were placed in a HS microwell plate in the
presence of imidazole at a final concentration of 5 mM. This is the
selective condition used for screening the different metal ion-affinity
clones. HS plates were incubated for 4 hours at room temperature. Plates
were then washed 3× with phosphate-buffered saline (PBS) containing
0.05% Tween 20 (PBS-T). The HS plates were then incubated with anti-GST
at 1:1,000 dilution in PBS-BSA buffer (0.2 ml/well) for 1 hour at room
temperature. HS plates were washed 3× with PBS-T. The HS plates
were then incubated with anti-mouse HRP conjugate at 1:10,000 dilution in
PBS-BSA buffer for 1 hour at room temperature. Plates were washed
3× with PBS-T. The plate was then developed with
2,2'azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABST substrate.
Color development was stopped by the addition of sodium azide to a final
concentration of 2 mM. Absorbance of the plates was read at 405 nm using
a Wallace 1420 plate reader. The HC plates were used to further analyze
potential clones. To further characterize the clones, 0.2 ml of cell
extracts were applied to the HC plates and the plates were incubated at
ambient temperature for 1 hour. The plates were washed with PBS as
described above. Twenty-one clones that produced the highest response on
the HS plates were eluted from the corresponding HC plate. The selected
cloned proteins were eluted from the HC plates by incubating at
37° C. for 15 minutes in 50 mM sodium phosphate, 0.3 M sodium
chloride and 0.2 M imidazole buffer. Eluted proteins were then moved to
clean tubes and analyzed by SDS-PAGE. All 21 clones had the expected
molecular weight and were sequence verified.

[0167] These 21 colonies were grown overnight in 1 ml LB/ampicillin media
at 37° C. at 250 rpm. 100 μl of the overnight cultures were
transferred to 50 ml of fresh LB/ampicillin media and the cultures grown
for an additional 3 hours at 37° C. The cultures were induced with
IPTG (final concentration of 1 mM) and the cultures grown for an
additional 3 hours prior to harvesting by centrifugation.

Example 2

Construction of a N-Terminal Metal Ion-Affinity Fusion Protein

[0168] Two metal ion-affinity tags were introduced to the N-terminal of
bacterial alkaline phosphatase (BAP). The constructs were constructed
from the BAP expression vector pFLAG-CTS-BAP. Construction was done by
annealing two pair of complimentary oligonucleotides together. The
following oligonucleotides were constructed:
5'TATGCATAATCATCGACATGAACATA3'(SEQ ID NO: 20),
5'AGCTTATGTTTATGTCGATGATTATGCA3' (SEQ ID NO: 21),
5'TATGCATAAACATAGACATGGGCATA3' (SEQ ID NO: 22) and
5'AGCTTGATGCCCATGTCTATGTTTATGCA3' (SEQ ID NO: 23). The oligonucleotides
were annealed together to generate a cassette. The cassette was ligated
into pFLAG-CTS-BAP, which had been digested with NdeI and HindIII
restriction endonucleases. Ligated vector was transformed into E. coli
DH5-a using standard protocols and plated on LB/ampicillin.

Example 3

Expression of an N-Terminal Metal Ion-Affinity Fusion Protein

[0169] MAT-BAP fusion peptide cultures were grown overnight in 1 ml
LB/ampicillin at 37° C. 500 μl of overnight cultures were
transferred to 500 ml of fresh TB media containing ampicillin (100 mg/L).
The cultures were grown for three hours at 37° C. at 250 rpm.
Protein expression was induced by the addition of IPTG (final
concentration of 1 mM). Cultures were then grown for an additional three
hours, harvested by centrifugation and stored at -70° C. until
further use.

Example 4

Metal Ion-Affinity Fusion Protein Purification Protocol #1

[0170] Cells were resuspended in 2 ml of TE (50 mM Tris-HCl pH 8.0, 2 mM
EDTA). Lysozyme (4 mg/ml in 2 ml of TE) was added to the resuspended
cells and the cells were lysed at ambient temperature for 4 hours. The
cell debris was removed by centrifugation at 27,000×g for 15
minutes. The supernatant was dialyzed overnight against 50 mM Tris-HCl pH
8.0 to remove the EDTA. The dialyzed supernatant was applied to a 1 ml
column containing a nickel bis-carboxy-methyl-cysteine resin (nickel
resin). The column was washed with 4 ml of 50 mM Tris-HCl pH 8.0 and then
washed with 2 ml of 50 mM Tris-HCl pH 8.0, 10 mM imidazole. The column
was then eluted 50 mM Tris-HCl pH 8.0 250 mM imidazole. Samples were
analyzed for purity by SDS-PAGE.

Example 5

Metal Ion-Affinity Fusion protein Purification Protocol #2

[0171] Cells were resuspended with CelLytic B (Sigma-Aldrich product no.
B3553), and 10 mM imidazole. The cells were solubilized by incubation for
15 minutes. The cell debris was removed by centrifugation at
15,000×g for 5 minutes at room temperature. A 0.5 ml column,
containing nickel resin, was equilibrated with 10 column volumes (5 ml)
of 50 mM sodium phosphate, pH 8, and 300 mM sodium chloride (column
buffer). The supernatant was loaded on the column. The column was washed
with 10 column volumes (5 ml) of 10 mM imidazole in column buffer. The
column was eluted with 100 mM imidazole in column buffer. The samples
were analyzed for specificity by SDS-PAGE.